Project Summary/Abstract Expression of the recently discovered human restriction factor SAMHD1 is responsible for the infection block imposed to lentiviruses such as HIV-1, HIV-2 and SIVmac by primary macrophages, dendritic cells and resting CD4+ T-cells. SAMHD1 blocks lentiviral infection by preventing the occurrence of reverse transcription. SAMHD1 has deoxynucleotide triphosphohydrolase (dNTPase) activity, which degrades deoxynucleotide triphosphates (dNTPs) into nucleotides and triphosphates, and this activity is required for HIV-1 restriction. However, additional studies have revealed that the dNTPase activity of SAMHD1 is not sufficient for HIV-1 restriction. Thus, an additional property of SAMHD1 is required for HIV-1 restriction. SAMHD1 interacts with nucleic acids in vitro, but the contribution of this interaction to HIV-1 restriction in vivo remains to be determined. Our preliminary data using SAMHD1 mutants indicate that nucleic acid binding is important for HIV-1 restriction. We determined the structure of SAMHD1 bound to an oligodeoxynucleotide and leveraged this structure as a tool for structure-function studies. Several amino acids form the interface between SAMHD1 and the oligodeoxynucleotide, and mutation of these residues resulted in SAMHD1 proteins that are unable to restrict HIV-1. These results suggest that the ability of SAMHD1 to interact with nucleic acids is important for HIV-1 restriction. Separate from its antiviral activity, mutations in the human SAMHD1 gene cause Aicardi- Goutires syndrome (AGS). AGS patients exhibit increased levels of type I interferon (IFN) that are believed to result from the recognition of endogenous nucleic acids by innate immune sensors. Thus, it has been suggested that the in vivo role of SAMHD1 is to prevent activation of the innate immune response by endogenous nucleic acids, and the nucleic acid binding activity of SAMHD1 has been suggested to be important for this function. Our preliminary data indicate that SAMHD1 knockout (KO) mice are resistant to herpes simplex virus 2 (HSV-2)-induced limb paralysis and death. These results suggest that SAMHD1 prevents the recognition of HSV-2 by innate immune sensors, potentially by interacting with the HSV-2 viral DNA and shielding it from detection. This strong phenotype in vivo will be used to unveil the role of SAMHD1 in innate immunity. Based on these published and preliminary results, the central hypothesis of this proposal is that the interaction of SAMHD1 with nucleic acids is important for its antiviral and innate immune functions. Our rationale is that identification of the mechanisms by which SAMHD1 modulates viral infection and innate immune responses will enable the development of novel antiviral and AGS therapies. To test our central hypothesis, we will pursue the following specific aims: 1) Determine the mechanism by which SAMHD1 inhibits HIV-1 infection, 2) Evaluate the role of SAMHD1 acetylation in SAMHD1-mediated viral restriction, and 3) Characterize the role of SAMHD1 in innate immunity.